Flow-induced forces resulting from intravital biofluids (e.g., blood, urine, cerebrospinal fluid) are widely acknowledged to be critical for proper embryonic development. Defects in embryonic biofluid flow are associated with renal, cardiovascular and nervous system disorders . Genetic, surgical, and pharmacological animal models exist or are being developed for both normal development and disease states. However, existing methods for intravital flow imaging are inadequate as all current modalities lack the spatial and/or temporal resolution necessary to describe the wide range of complex developmental flows that exist in biological organisms. We propose to create a cutting-edge, cross-platform technology for 4-D imaging (3-D + time) of biofluid flows within the living embryonic zebrafish, a widely-used animal model for developmental studies. The foundation of this critically-needed technology will be a laser-based multidimensional microscopic imaging system to visualize and track the motions of submicron fluorescent tracer particles suspended within the anatomical flows of interest. We will utilize a unique defocusing digital particle image velocimetry (DDPIV) technique to obtain the required spatial and temporal sensitivity. In addition, by developing new image analysis algorithms we will, for the first time, be able to account for the large velocity gradients and moving boundaries that are so prevalent in living systems and which have been so problematic for existing in vivo imaging technologies. The creation of a novel in vivo micro-DDPIV technology will greatly impact our ability to understand how biofluid flow affects development in both healthy and flow-compromised animals. This is an area of great need as we are currently capable of creating large numbers of flow-related mutants in zebrafish, but are far less able to reliably quantify the resulting dynamic flow changes in order to understand their effects on developmental pathogenesis. This work will significantly aid a wide-range of biomedical research efforts, as flow-dependent phenomena are key factors in a great many diseases including polycystic kidney disease, atherosclerosis, syringomyelia, cardiomyopathy, and hydrocephalus-related disorders.